Arbuscular mycorrhiza (AM) is a symbiosis between plants and AM fungi that requires the intracellular accommodation of the fungal partner in the host. For reciprocal nutrient exchange, AM fungi form intracellular arbuscules that are surrounded by the peri-arbuscular membrane. This membrane, together with the fungal plasma membrane, and the space in between, constitute the symbiotic interface, over which nutrients are exchanged. Intracellular establishment of AM fungi requires the VAPYRIN protein which is induced in colonized cells, and which localizes to numerous small mobile structures of unknown identity (Vapyrin-bodies). In order to characterize the identity and function of the Vapyrin-bodies we pursued a dual strategy. First, we co-expressed fluorescently tagged VAPYRIN with a range of subcellular marker proteins, and secondly, we employed biochemical tools to identify interacting partner proteins of VAPYRIN. As an important tool for the quantitative analysis of confocal microscopic data sets from co-expression of fluorescent proteins, we developed a semi-automated image analysis pipeline that allows for precise spatio-temporal quantification of protein co-localization and of the dynamics of organelle association from movies. Taken together, these experiments revealed that Vapyrin-bodies have an endosomal identity with trans-Golgi features, and that VAPYRIN interacts with a symbiotic R-SNARE of the VAMP721 family, that localizes to the same compartment. Arbuscular mycorrhiza (AM) represents a wide-spread symbiotic association of plants with a monophyletic group of fungal endosymbionts (AM fungi) that are collectively known as the Glomeromycotina in the order Mucuromycota  Forward genetic screens to identify genes required for symbiosis, and (ii) transcript profiling to identify AM-induced genes followed by reverse genetic analysis of their function by gene knockouts or gene silencing. The first approach has been very successful, in particular to identify genes required for early symbiotic signaling  domain [also known as major sperm protein (MSP) domain], and a C-terminal ankyrin (ANK) domain with 11 ankyrin repeats  with Agrobacterium tumefaciens (agro-infiltration) is an established procedure for transient gene expression and subcellular localization studies   together with VAPYRIN-GFP (Wave 3R) , which encodes a SNARE protein that specifically localizes to Golgi stacks . Indeed, significant co-localization was observed (TGN elements converge with recycling endosomes (RE) in anterograde trafficking from the Golgi  and RabF2a (Wave 7R) highlight multivesicular bodies (MVB), late endosomes, and prevacuolar compartments   , that haave 11R) . In addiave 11R) , and whiWave 138) , which localizes to autophagosomes  . Taken thttp://www.kalaimoscope.com/science.html). This tool uses specialized algorithms to process movies, involving global object recognition, establishment of movement tracks over time, and quantification of various parameters related to object area and movement tracks. Quantification from movies by movement tracks allows more consistent analysis than assessment of individual images. We used this tool to quantify co-localization and object association with a range of Wave markers and additional subcellular marker proteins.Characterization of subcellular compartments by protein co-localization studies requires detailed spatio-temporal image analysis. Selection of representative images and counting of co-localizing structures by visual inspection may not always be sufficient for a quantitative analysis of such phenomena. In addition, individual images cannot reveal dynamic aspects that can only be observed in movies. Hence, we developed a semi-automated bioimage-informatics pipeline based on the Kalaimoscope MotionTracker software  yielded an average distance between the two channels of 0.12 ± 0.07 μm (median = 0.11 μm) . Taken together, these results suggest that Vapyrin-bodies have an identity that involves features of recycling endosomes and TGN, and that they are associated with the Golgi apparatus.Besides MEMB12, which exhibited association rather than co-localization with Vapyrin-bodies , S3, two0.11 μm) . These s0.11 μm) . Studentee pairs  were higN. benthamiana, or in the native context, namely in P. hybrida stably transformed by Agrobacterium tumefaciens through leaf disc transformation, and in hairy roots of P. hybrida transformed by Agrobacterium rhizogenes . Thus, VAPYRIN-GFP and VAP-GFP were expressed either transiently in In general, expression of VAPYRIN-GFP and VAP-GFP was always observed at low levels, although the constitutive cauliflower mosaic viral 35S-promoter was used. In addition to the low levels of expression, part of the fluorescent signal corresponded to free GFP . This suThe central issue in solubilizing protein complexes for CoIP and MS analysis is to solubilize the complexes with detergents without interfering with the protein:protein interactions in the complex. A number of protocols have been established for receptors and other membrane proteins to achieve this goal . A total of 324 clones were recovered from a primary screen on 3.2 × 106 clones , M. truncatula (Mt), L. japonicus (Lj), rice , and A. thaliana (At) revealed that VAMP721m, together with 3 additional paralogs, (VAMP721x through VAMP721z) falls into a clade with the symbiotic VAMPs from M. truncatula (MtVAMP721d and MtVAMP721e)  ; File S1AMP721e) . This clAlthough Vapyrin-bodies exhibited RFP-VAMP721m signal , an evenN. benthamiana proved rather difficult. In most cases, agro-infiltration experiments produced very few cells that co-expressed both markers, whereas this method worked routinely with other fluorescent markers (see above). These results suggest that the co-expression of VAPYRIN and VAMP721m at elevated levels is detrimental to tobacco leaf cells. As a consequence of the problems with co-expression and the resulting deficit in image quality, the imaging results from confocal microscopy of VAPYRIN-GFP and RFP-VAMP721m were not suitable for processing with the MotionTracker quantification pipeline.It should be noted here that co-expression of VAPYRIN-GFP and RFP-VAMP721m in More direct information on protein:protein interactions can be obtained with bi-molecular fluorescence complementation (BiFC). BiFC is based on the reconstitution of YFP fluorescence from two split halves that are brought together by the interaction of two fused proteins of interest . Both exhibited distinct Vapyrin-bodies, however, in Arabidopsis roots, they were immobile, suggesting that Arabidopsis has subcellular compartments similar to Vapyrin-bodies, but lacks a trafficking component required for their active translocation. In contrast, tobacco leaves exhibited Vapyrin-bodies with a similar behavior as in P. hybrida roots  screen with the VAP-domain as a bait. With this strategy, we identified a vesicle-associated membrane protein (VAMP721m) that co-localized with VAPYRIN . PhylogeVAMP721e , which fVAMP721e . As in tnization . This isnization  may requnization , or SYP1nization .M. truncatula, VAPYRIN and EXO70I have been shown to be located in the vicinity of the peri-arbuscular membrane (PAM) around the tips of branching hyphae , followed by 7% bleach with 0.1% Tween 20, and rinsed 5 times before sawing to seedling substrate (Klasman). After 1 week from germination, plantlets were transferred to single pots with clay substrate (Klasman). Once a week soil was treated with iron fertilizer (Optifer from Optima). Temperatures in growth chambers were adjusted to 32°C during the light phase (12 h) and 22°C during the dark phase (12 h). For the production of sterile in vitro plant material for transformation . The culture was then centrifuged at 4,000 g for 15 min at room temperature. The pellet was resuspended and adjusted to an OD600 of 0.7 in AS medium . Each culture was mixed 1:1 with a culture expressing the viral repressor p19 , and signal was acquired between 500 and 550 nm. For mCherry and mRFP, a helium-neon laser was employed for excitation (543 nm), and fluorescence was acquired between 590 and 640 nm. Confocal images were analyzed with the Leica LAS AF Lite and FIJI/ImageJ softwares. All double-channel analyses were acquired in the sequential mode to avoid “bleeding-through” between the channels. For the analysis of the dynamic behavior of Vapyrin-bodies, and for quantitative analysis of colocalization and association (see below), time series (movies) were acquired. For each construct, at least three independent transformation experiments were conducted, each with several plants of which several leaves were infiltrated. Representative movies were chosen for quantitative analysis.Transient expression assays were performed with three to 5-week-old plants as described . This software identifies elliptic objects by fitting Lorenzian functions to them according to user-defined object-search parameters. This allows to determine the x-y position with sub-pixel accuracy, as well as further object properties including size, area, intensity and position. Once the object search is complete for all the consecutive frames of a time-series (movie) in two channels, MotionTracker can then search for tracks. To this end, its algorithm searches the most probable association between objects in adjacent frames of the two channels, and establishes the path of all objects during the time-series. Object size, speed and direction are calculated to determine the object tracks. The visualization of the object tracks by plotting their path as an overlay on the image sequence allows easy visual inspection of the object tracking results to confirm their accuracy and, if necessary, to adjust the tracks manually.Quantification of object co-localization and association was performed with a semi-automated image processing pipeline based on the Kalaimoscope MotionTracker software version 8.88.15  , left. WIn order to identify interacting partners of VAPYRIN, the split-ubiquitin Y2H screen from DUALhunter was employed . All procedures were carried out as detailed in the manufacturers protocol . To prepare the bait vector, the VAP domain was amplified by PCR from cDNA prepared from RNA extracted from mycorrhizal petunia roots using the following primers:Forward primer:5′-GAGTGGCCATTACGGCCATGGATAGACTATTAAGCTTAGAGCCATC-3′; Reverse primer:5′-TCGACATGGCCGAGGCGGCCGTAGCTCCAGGGGCTACAAC-3′.Saccharomyces cerevisiae reporter strain NMY51 (kindly provided by Claudio de Virgilio) by electroporation according to the manufacturers protocol. S. cerevisiae NMY51 is auxotrophic for tryptophan, leucine, histidine, and adenine . All procedures were carried out according to the recommendations of the provider.Both primers contained asymmetric SfiI restriction sites to allow directional cloning into the SfiI sites of the pDHB1 vector. The amplicon was cloned into pGEM®T-easy , sequenced and recovered by SfiI digestion . It was then ligated into pDHB1 to obtain the pDHB1-VAP bait vector and transformed into R. irregularis MUCL 43,204 was used for cDNA synthesis. Before reverse transcription, mRNA was enriched with Oligotex Direct mRNA Mini Kit . The first strand cDNA was synthesized by the EasyClone reverse transcriptase (RT) using a 3′-end adapter containing an SfiI site and oligo (dT). This RT reaction adds oligo (dC)s to the 5′-end of the first strand, allowing the 5′-end adapter that contains the second SfiI site to bind. An evaluative PCR using 1 μl of the first strand cDNA was performed to determine the optimal number of cycles for PCR amplification. Based on this test, the second strand was amplified by PCR from the first strand cDNA with 17 cycles, using primers containing SfiI sites A and B. DNAs were purified with the NucleoSpin Extract II kit  and digested with 2 μl SfiI restriction enzyme during an overnight incubation at 50°C. cDNAs longer than 400 bp were selected by size fractionation . Note that in contradiction to the recommendations of the provider, cDNAs were collected in the third elution, which was controlled by gel electrophoresis. cDNAs were purified by phenol/chloroform extraction and quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The cDNA was then directionally inserted downstream of NubG using two asymmetric SfiI restriction sites. A test transformation in E. coli Top10 was performed to check for the number of independent transformants in the final library. To test the quality of the library, 10 randomly picked colonies were analyzed by colony PCR with pPR3N primers.The cDNA library (prey vectors) was constructed with the EasyClone cDNA Library Construction Kit , according to the provider's recommendations. In brief, 5 μg total RNA extracted from a 50:50 mixture of non-colonized roots and mycorrhizal roots colonized by To optimize the stringency of the screen, a first pilot screen was carried out that simulated the conditions of a library screen, but with the empty library vector (no bait) instead of the cDNA library vectors. NMY51 cells expressing DHB1-VAP were pre-cultured in liquid SD-Leu, then grown to an OD600 of 0.6–0.8 in 2x YPAD, and transformed with pPR3-N  using a high-efficiency library scale transformation. For that, they were incubated 45 min. at 30°C with the master mix and the plasmids and then heatshocked for 20 min. at 42°C with DMSO. Transformed yeast was re-suspended in 2x YPAD and incubated 90 min. at 30°C with slow shaking for recovery. The cells were then resuspended in 0.9% NaCl and plated on different media of increasing stringency: (i) SD-2D: three 100 mm ø plates, to test for transformation efficiency. (ii) SD-3D: six 150 mm ø plates lacking W, L and H, each supplemented with an increasing concentration of 3-aminotriazole (3-AT): 0, 1, 2.5, 5, 7.5, 10 mM. (iii) SD-4D: six 150 mm plates lacking W, L, H, and A, containing the same concentration of 3-AT as SD-3D plates. Plates were then incubated at 30°C and yeast growth was assessed. VAP bait should not interact with the empty library vector . HoweverVAP-expressing NMY51 was transformed with 28 μl library plasmid with a high-efficiency transformation method (see above). Transformants were plated on SD-2D plates to test for transformation efficiency and on SD-4D without 3-AT, because this condition was establishes as 100% selective. Positive clones where recovered and cultured on SD-2D plates for the X-Gal filter assay. For further characterization, DNA from yeast clones with putative interactors was extracted and transformed into XL1-blue. Plasmid DNA from transformed bacteria was extracted with ZR miniprep kit and tested for candidate cDNA insert by digestion with SfiI.Interaction strength and specificity with the VAP domain was determined by a drop test. Candidate clones were re-transformed into NMY51expressing the bait VAP fusion protein. Transformed yeast was cultured in 2 ml selective medium (SD-2D) overnight at 30°C, adjusted to an OD600 of 0.2 and re-grow to an OD600 of 0.8–3. 200 μl of each culture was diluted to an OD600 of 1 in a microtiterplate in 4 dilution series: 1/1, 1/10, 1/100, 1/1000. Sterile water was used for the dilutions. 3 μl of each dilution were spotted onto 2 plates in parallel: SD-2D, to test for the presence of the plasmids and the cell number of a drop and SD-4D, to test for protein-protein interaction of the bait and prey protein pairs. After 24 h at 30°C, colonies started to be visible on SD-2D plates. After 36–48 h, growth was observed on SD-4D plates. Pictures were taken after 36 h for SD-2D plates and after 3 days for SD-4D plates. Control vectors were used for comparison in combination with pDHB1-VAP; pA1-Alg4 as a positive control and pPR3-N (EV) as a negative control of interaction. Additionally, each candidate clone was also tested with the empty bait vector  by a drop test to test for specificity of the interaction with the VAP domain. Prey candidates interacting strongly with the empty bait were eliminated, the other candidates were considered true interactors of VAP. Additional information concerning the biochemical and genetic search for interactors of VAPYRIN can be found in the Supplementary Materials and Methods .www.phylogeny.fr  required for the establishment of arbuscular mycorrhiza.All datasets generated for this study are included in the manuscript and/or the LB, SL, GD, CM, NF, and MC conducted experiments. GD and FM analyzed microscopic data. DR conceived the project and wrote the manuscript with the help of all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Furthermore, we found that by treating the cytokinin nucleotides with alkaline phosphatase prior to analysis, the sensitivity of the detection process could be increased. In addition, derivatization (propionylation) improved the ESI-MS response by increasing the analytes' hydrophobicity. Indeed, the ESI-MS response of propionylated isopentenyladenosine was about 34% higher than that of its underivatized counterpart. Moreover, the response of the derivatized zeatin ribosides was about 75% higher than that of underivatized zeatin ribosides. Finally, we created a web-based calculator (IZOTOP) that facilitates MS/MS data processing and offer it freely to the research community.An improved method for determining the relative biosynthetic rate of isoprenoid cytokinins has been developed. A set of 11 relevant isoprenoid cytokinins, including zeatin isomers, was separated by ultra performance liquid chromatography in less than 6 min. The iP-type cytokinins were observed to give rise to a previously-unknown fragment at  N-glucosides) or at the hydroxylated side chain (O-glucosides). However, only the free bases are thought to have significant biological activity. Accordingly, the interaction between the cytokinin ligand and its receptor is strongly influenced by the character of the side-chain. Nevertheless, cytokinin-receptor assays have shown that at least some receptors also respond to nucleosides and nucleotides [The cytokinins are a group of plant hormones that play a central role in the regulation of cell division and differentiation. They control processes as diverse as apical dominance, root formation, leaf senescence, stomatal behavior, and chloroplast development . Althougleotides .N-prenylation of adenosine 5’-phosphates  with dimethylallyl diphosphate or hydroxymethylbutenyl diphosphate as side-chain donor - is catalyzed by the appropriate isopentenyltransferase . The substrate specificity of IPTs varies depending on their origin and the species from which they are derived. In Arabidopsis thaliana, IPTs predominantly use ADP or ATP rather than AMP as prenyl acceptors, forming isopentenyladenosine 5’-diphosphate and 5’-triphosphate [trans-hydroxylated by cytochrome P450 monooxygenases to form trans-zeatin-type nucleotides [The first dedicated step in cytokinin biosynthesis - hosphate ,5. The ileotides . In addiPlant tissue extracts are complex multi-component mixtures that contain cytokinins in minute quantities (fmol to pmol per gram of fresh weight) along with other compounds with similar structures and/or physico-chemical properties. Therefore, mapping the cytokinin composition of plants requires the use of sensitive and highly selective analytical methods. Hyphenated techniques such as ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) have revolutionized trace analysis of cytokinins . These min vivo deuterium labeling [2O, deuterium was incorporated into general metabolic pathways and newly synthesized molecules thus labeled without altering their steady state levels [The most common method to assess biosynthetic activity in biological systems involves the use of labeled precursors. Previously, both radioactive and stable-isotope precursors have been applied to elucidate cytokinin biosynthesis in various plant species ,10. Becalabeling . By growe levels . The cyte levels . A modife levels . In the PGA22, and the results so obtained were compared to those obtained using an alternative method based on the analysis of underivatized cytokinins. Finally, we created a web-based calculator to process mass spectrometric data and convert them to tracer:tracee ratios (t/t), as described by Åstot et al. [In addition, by treating the cytokinin nucleotides with alkaline phosphatase followed by immunoaffinity purification of the dephosphorylated ribosides, we increased the sensitivity of the mass spectrometric detection. This method was used to measure the rate of synthesis of selected isoprenoid cytokinins isolated from the cytokinin‑overproducing Arabidopsis line t et al. .As analytical methods evolve, it becomes necessary to perform increasing numbers of separations. To make it easier to cope with this growing need, we sought to improve on the current HPLC method for the separation of propionylated cytokinins . GeneralOn the basis of this experience, we sought to develop a UPLC method using the same 50 mm column for the separation of propionylated cytokinins. A gradient elution program was designed using the Acquity UPLC columns calculator  and slightly adjusted to achieve a baseline separation of 11 isoprenoid cytokinins, including free bases, nucleosides and nucleotides. The HPLC method previously used for such separations employed a 150 mm column and took 56 min; the new UPLC method uses a 50 mm column and has a chromatographic separation time of less than 6 min. The gradient elution buffers used in this method (water and acetonitrile) typically contain 3% of added formic acid , which cm/z 69 in the spectra of various iP-type cytokinins, including the free bases, ribotides, ribosides and glucosides . Moreover, the fragmentation patterns were consistent with previously acquired FAB MS spectra . Howeverucosides .6-iP) by quadrupole-time-of-flight mass spectrometry. We reasoned that if unlabelled iP gives a side-chain fragmnent at m/z 69, D6-iP should give rise to an analogous fragment at m/z 75 due to the incorporation of six deuterons into the terminal methyl groups of the isopentenyl moiety. The results obtained are summarized in To confirm the identity of this fragment we performed an exact mass determination of the fragments of isopentenyladenine (iP) and its deuterium-labeled counterpart  [m/z 504 as the precursor ion and the free base fragment at m/z 204 as the product ion for multiple reaction monitoring of isotopomer I0 (the isotopomer with lowest m/z), to exclude the signal of the ribose moiety. The isopentenyl moiety can be trans-hydroxylated by cytochrome P450 monooxygenases to form trans-zeatin-type nucleotides [m/z 576-276 diagnostic transition for the I0 isotopomer. This transition corresponds to the dissociation of pro-zeatin riboside to its aglycone. An overview of the diagnostic transitions examined in this work is given in The nthesis) . Labelinnthesis) ,5, we seleotides . The rati.e. mono-, di-, and tri-phosphates), making it possible to analyze all of them at once; while this approach is obviously less informative than an analysis of the individual nucleotides, it is very convenient and efficient. We recently published a HPLC-MS method for the determination of underivatized intact cytokinin nucleotides in human leukemia cells [Generally, cytokinin nucleotides can be analyzed by LC-MS either as intact monophosphates ,17 or asia cells . Howeveria cells ,17. It ipga22, which carries the AtIPT8 gene under the control of the 17-β-estradiol-inducible promoter/enhancer [i.e. increased ratios of labeled to unlabeled cytokinin after correcting for the natural isotope distribution .As expected, induction of AtIPT8 expression triggered an increase in the biosynthetic rate. The iP-type nucleotides (iPNP) were more extensively labeled than were the tZ-type nucleotides (tZNP) throughout the experiment. This is consistent with iPNPs being the primary products of IPT action and tZNP being synthesized from them by trans-hydroxylation. In addition, both ribosides were less extensively labeled than their phosphorylated counterparts, confirming that n system . The tram/z region with relatively little chemical noise, resulting in a slightly higher MS-response. The sensitivity of MS detection is very important when designing specific biological experiments. Previously, at least 1 g of fresh plant tissue was required for measurements of the cytokinin biosynthetic rate [Finally, we compared the newly-developed UPLC-MS/MS method to that developed by Dobrev and co-workers,  which watic rate , which itic rate . Althougpga22 line by both methods. Cytokinins were isolated by harvesting Arabidopsis seedlings 6 hours after chemical induction of AtIPT8, fractionating them to separate the ribosides from the nucleotides, treating the latter with AP, and finally purifying them by IAC. The results are summarized in Both approaches, with or without derivatization, can be used to measure the relative biosynthetic rate of cytokinins. In both cases, analysis of peaks corresponding to the loss of the ribose moiety should yield similar results. To confirm this assumption we measured the relative biosynthetic rates of iPNP and iPR in the in vacuo). Nonetheless, this step is worthwhile because it greatly increases the sensitivity of the mass spectrometric detection. Since all of the relevant cytokinin metabolites are well resolved by UPLC, it is possible to analyze the whole set at once if necessary. The method desribed in this paper could potentially be used to analyze deuterium enrichment in all of the various kinds of cytokinins, including nucleotides, ribosides, free bases and glucosides, provided that one could obtain enough material. In this work, we decided to study the rather small plant Arabidopsis thaliana, which is a very popular model organism in plant sciences. However, due to the low mass of the plant seedlings and the relatively low levels of physiologically active CK bases, we chose to focus exclusively on nucleotides and ribosides in this instance.As expected, no significant differences were observed in the relative biosynthetic rates of iP-type cytokinins. Both UPLC-MS/MS methods are rapid, achieving complete separation in less than 6 min. Sample preparation remains the most time consuming part of the entire method; the derivatization in particular requires at least 10 hours .We have also created a web-based calculator (IZOTOP), which helps the user to convert signals obtained by MS/MS measurements into relative biosynthetic rate (t/t ratio) values. The procedure used in performing these calculations was adapted from the work of Åstot et al. . This caAuthentic cytokinin standards were purchased from Olchemim , formic acid from Merck , and DEAE-Sephadex A-25 from GE Healthcare . Deionised (Milli-Q) water was obtained from a Simplicity 185 water system . All other chemicals were of analytical grade or higher purity and were purchased from Sigma-Aldrich Chemie . Arabidopsis thaliana line pga22 carrying the AtIPT8 gene under the control of the 17-β-estradiol-inducible promoter/enhancer [enhancer  was growet al. [Samples were extracted and purified essentially as outlined by Novák et al. . BrieflyTM mass spectrometer (Waters MS Technologies) was used for the high-resolution identification and confirmation of the side-chain fragments at m/z 69 and m/z 75. Electrospray ionization in the positive ion mode was used with the following parameters: source block/desolvation temperature, 100 °C/350 °C; capillary/cone voltage, 2500/25 V; and spray/cone gas flow (N2), 500/50 L/h. In the product ion scan of the selected precursors (m/z 204 and m/z 210), data were acquired in the mass range m/z 50–250, with a cycle time of 33 ms, a scan time of 2.0 s, and collision energy of 40 eV, 50 eV and 60 eV. For the exact mass determination experiments, a lock spray was used for external calibration with a mixture of 0.1 M NaOH/10% formic acid (v/v) and acetonitrile  as a reference. Accurate masses were calculated and used for the determination of the elementary composition of the analytes with fidelity of 5 ppm.A hybrid Q-TOF microAn Acquity UPLC™ System  coupled to a Xevo triple-stage quadrupole mass spectrometer  was utilized for the cytokinin measurements. The system was controlled by Masslynx software .-1, the following elution profile was used: an isocratic step at 15% B for 0.5 min preceded a linear gradient from 15 to 45% B for 5.5 min. At the end of the gradient there was a washing step at 100% B (for 2 min) and then the column was equilibrated to the initial conditions for 1.9 min. The column was thermostated at 40 °C. Tandem mass spectra of all cytokinins examined in this study were acquired by continuous infusion of 10-3 mol L-1 solution in solvent A/solvent B  at a flow-rate of 15 μL min-1.The capillary voltage, cone voltage, collision cell energy, and ion source temperature were optimized for each individual compound. The mass spectrometer settings were as follows: a capillary voltage of 3 kV, a cone voltage of 33 V, a source temperature of 120 °C, a desolvation temperature of 550 °C, a cone gas flow of 70 L h-1, a desolvation gas flow of 600 L h-1, collision cell energy of 19 eV, and a collision gas flow of 0.2 mL min-1.Derivatized cytokinins were separated on a chromatographic column  using 0.7% HCOOH in water and 0.7% HCOOH in acetonitrile as solvents A and B, respectively. At a flow-rate of 0.5 mL min4OH) and methanol as solvent A and B, respectively. At a flow-rate of 0.5 mL min-1, the following elution profile was used: an isocratic step at 10% B for 0.5 min preceded a linear gradient from 12 to 45% B for 3.5 min. At the end of the gradient there was a washing step at 100% B (for 0.3 min) and then the column was equilibrated to the initial conditions for 1.2 min. The column was thermostated at 40 °C. The capillary voltage, cone voltage, collision cell energy, and ion source temperature were optimized for each individual compound. The mass spectrometer settings were as follows: a capillary voltage of 3 kV, a cone voltage of 30 V, a source temperature of 120 °C, a desolvation temperature of 575 °C, a cone gas flow of 70 L h-1, a desolvation gas flow of 600 L h-1, collision cell energy of 19 eV, and a collision gas flow of 0.2 mL min-1.Underivatized cytokinins were separated on chromatographic column  using 15 mM HCOOH  that facilitates MS/MS data processing and have made it freely available to the research community.


Due to the effect of severe environmental conditions, such as intense heat, blowing sand, and ultraviolet light, conventional pesticide applications have repeatedly failed to adequately control mosquito and sandfly populations in desert areas. In this study, a vinyl silsesquioxane (VS) was added to a pesticide  to enhance residual, thermal and anti-ultraviolet properties via three double-bond reactions in the presence of an initiator: (1) the connection of VS and citral, (2) a radical self-polymerization of VS and (3) a radical self-polymerization of citral. VS-citral, the expected and main product of the copolymerization of VS and citral, was characterized using standard spectrum techniques. The molecular consequences of the free radical polymerization were analyzed by MALDITOF spectrometry. Anti-ultraviolet and thermal stability properties of the VS-citral system were tested using scanning spectrophotometry (SSP) and thermogravimetric analysis (TGA). The repellency of VS-citral decreased over time, from 97.63% at 0 h to 72.98% at 1 h and 60.0% at 2 h, as did the repellency of citral, from 89.56% at 0 h to 62.73% at 1 h and 50.95% at 2 h. Mosquitoes are important vectors of arthropod-borne viruses such as yellow, dengue and chikungunya fevers, causing worldwide outbreaks of severe epidemic diseases every year . ResearcIn recent years, the growing interest in polyhedral oligomeric silsesquioxanes (POSS) or silsesquioxanes (SSO) is evidenced by the increased number of studies published and from both experimental and theoretical investigations due to the unique physical and chemical properties of silsesquioxanes ,16,17,18Due to the absence of VOC in SSO materials, these systems possess inherently low flammability and thus are not subject to uncontrolled polymerization . There aVinylsilsesquioxanes ,25 and eCommercial vinyltrimethoxysilane  was used as a precursor in the study of hydrolytic condensation to prepare vinyl-SSO, ethanoland tetrahydrofuran (THF) were used as solvents (99.7% purity) and formic acid was employed as a catalyst (88% purity); citral  can be selected as a main component of the modified pesticide; sodium dodecyl sulfate (SDS) as a surfactant; and potassium persulfate (PP) as an initiator were supported by Tianjin BoDi Chemicals and Tianjin Fine Chemicals , respectively. All of the above reagents are of analytical purity, used without further treatment.For the hydrolytic condensation reaction, VMS was placed in beakers. Ethanol was added to give a 3:1 molar ratio with respect to Si. The polycondensation was carried out in the presence of formic acid, added in a 0.5:1 molar ratio with respect to Si. The corresponding molar ratio of water to Si was 1:1. The beaker was sealed with a plastic film and left for a day at 35 °C. Then needle-sized holes were made in the plastic film and the reaction was continued for another day at the same temperature. Finally, the plastic film was removed and the reaction continued for one more day at 65 °C. The reaction products were homogeneous viscous liquids that could be completely dissolved in different solvents such as tetrahydrofuran (THF), chloroform and ethanol. The SSO based on VMS will be denoted as VS (VMS-SSO).The emulsion polymerization was employed to prepare the compound of the pesticide modified with VS as shown in The structures of the VS, citral and VS-citral were characterized using Fourier transform infrared spectroscopy  and Biflex III matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry , and the equipment measuring conditions of UV-MALDI-TOF MS for characterization of the VP-pesticides in detail were similar to that in the previous work ,29.−1 from 0 to 800 °C.For the properties, a UV-3101PC Scanning Spectrophotometer  device was used to measure the film transparency in a range of wavelengths between 280 and 400 nm (UV-A and UV-B region). TGA was performed on TA Instruments ZRY-2P equipment  under nitrogen atmosphere at a heating rate of 20 °C·minAedes albopictus  mosquitoes were used for repellent testing. Mosquito larvae were obtained from the Department of Entomology of Chinese Center for Disease Control and Prevention . The larvae were reared at 28 °C and 60% relative humidity. Adult mosquitoes were fed and maintained on a 10% sucrose solution. Two kinds of mosquito repellents, 5% citral , and 5% VS-citral, were purchased and prepared. Aliquots of 1.5 mL were applied to volunteers’ forearms to test repellent efficacy n(OH)x(OCH3)y  (as seen in m/z) and the calculated molecular weight according to ion adducts (M + H+ or M + Na+ or M + K+) [ M + K+) ,39,40.Tn clusters in 5 and T6 clusters, each larger frame contains two main peaks. One part of the peak data in the small square frames for T7–T13 clusters  and ultraviolet-A (UV-A) region (280–400 nm) see , the aveThermogravimetric analysis shows that there is about 15% citral released from VS-citral. Citral and VS-citral were tested on eight volunteers each. Botanical mosquito repellents such as citral and VS-citral according to the China Food and Drug Administration (CFDA) guideline. Repellency tests of 5% citral were performed on 8 volunteers. A mean (±SE) of 23.38 ± 0.66 mosquitoes landed on their untreated left forearms during exposure to 300 mosquitoes for 3 min . RepelleTn sub-clusters in the main Tn cluster: one Tn sub-cluster indicated a normal Tn cluster with three citrals and the other sub-cluster indicated that one citral among three in the normal Tn cluster deleted an enolic fragment (mass: 44) after the McLafferty Rearrangement.A pesticide  can be modified with VS to enhance anti-ultraviolet and thermal stability in desert areas. During the modification, there were three double-bond reactions in the blend of citral and VS in the presence of an initiator. The product of the main reaction  was characterized by FTIR and MS spectra. From the MS spectrum, there were two The measured anti-ultraviolet and thermal stability of the VS-citral oligomer gives it great potential as a modifier to improve the properties of pesticides due to the SSO peculiar nanometer structure.We have successfully prepared a silsesquioxane-based delivery system for citral that contains more than 15% citral for slow release to provide long-term protection against insect pests. The material is easy to prepare from commercially available VMS, a common coupling agent, and citral, a common terpene, essential oil. This insect repellent/pesticide should provide a cost-effective alternative to synthetic chemical-based systems.No work had been conducted in search of insecticidal activity of citral and VS-citral. Citral and VS-citral were for the first time evaluated as mosquito repellent and could make excellent acting insecticide, but perhaps their greatest strength would be their use as an insect repellent.


We found that antioxidant hierarchies depended on the choice of assay and applied ANOVA analyses to explore underlying structure-TAC dependencies. In addition to statistically confirming the empirically established connection between flavonoid ring-B catechol and high TEAC or FRAP, new correlations were also found. In flavonoids, (i) hydroxyl groups on ring-B had a positive effect on all four TAC assays; (ii) the presence of a 3-hydroxyl group on ring-C increased TEAC and FRAP, but had no effect on DPPH or FCR; (iii) Phenolic acids lacking a 3-hydroxyl group had significantly lower FRAP or DPPH than compounds having this structure, while TEAC or FCR were not affected. Results demonstrated that any TAC-based ranking of phenolic rich samples would very much depend on the choice of assay, and argue for use of more than one technique. As an illustration, we compared results of the above four assays using either grapevine leaf extracts or synthetic mixtures of compounds prepared according to major polyphenols identified in the leaves.Thirty-seven samples of naturally occurring phenolic compounds were evaluated using three common  There are two mechanisms for the radical scavenging reactions of phenolic antioxidants: a hydrogen atom transfer from the phenolic OH group; and an electron transfer followed by a proton transfer. The most frequently used free radicals are 2,2-diphenyl-1-picrylhydrazyl  and theyl . This pet al. . In anot samples .The aim of the present study was to investigate how critical is the choice of TAC method when evaluating polyphenol rich samples. We chose four popular assays: TEAC, FRAP, DPPH, and FCR, and compared results over a set of 37  pure test compounds. We believe that this is the first work that shows a comparison of all four techniques and examines possible redundancies. Interchangeability of assays would assume that different techniques assigned higher TAC to compounds belonging to the same polyphenol group. Therefore, we also examined how chemical structures influenced assay results. Structure-activity relationships have already been addressed in a number of publications reporting TAC properties of pure compounds, but these works were either focused on one method, such as TEAC ,27,28,29The obtained data set allowed us to address to which extent TAC values of pure test compounds are relevant to antioxidant capacities of plant extracts containing these compounds, for example whether structurally different compounds can lead to an additive TAC in plants. Reports on this issue are contradictory, including both antagonistic and synergistic effects when antioxidants are combined ,34. UsinPhenolic compounds used in this study are listed in et al. [To compare results of various TAC assays, these have to be uniformly calibrated by using the same reference compound. Results of TEAC, FRAP or DPPH assays are usually calibrated using ASA or Trolox, and FCR is traditionally calibrated with gallic acid. The choice of reference compounds has been studied and discussed extensively by Nenadis et al.  concludiO-glucoside was the most suitable reference compound for the above requirements, because it gave very similar raw data in the TEAC, FRAP and DPPH assays . Among the studied compounds, myricetin-3-In order to evaluate whether TAC assays favored any compound groups, we made pair wise graphical comparisons . In FiguFor example, among flavonol glycosides (represented by filled black circles in R2), all linear regressions were statistically significant at p < 0.001. Although its TAC nature is well-recognized [cognized , FCR is In the following, the two major phenolic subgroups, flavonoids and phenolic acids, are analyzed separately. There is a well-established hierarchy of flavonoids in terms of chemical structure and antioxidant capacities. However, these studies are usually concentrated on one method only. TEAC ,15,44, Det al. [p < 1% significantly coupled to high TAC values of all four methods  making the testing of individual effects unreliable.Ring-C 2,3 double bond in conjugation 4-oxo function was reported to enhance TEAC , althouget al. . We testO-glycosides. Glycosylated flavonoids were found to have lower TEAC [p < 1% . The striking difference between DPPH assay results of kaempferol and kaempferol-glycosides have already been observed [O-glycosides as compared to their aglycones : the glycosylated flavanone had a 10% higher TEAC than its aglycone form. There was no difference in the FCR of these two compounds—glycosylation lowered both FRAP and DPPH by 45% and 80%, respectively. The difference between the effect of glycosylation on TEAC in flavonols and hesperitin could be explained by differences in ring-C structures, but is more likely due to the fact that hesperidin was glycosylated at its A-ring 7-O position and not at the C-ring 3-O position as all flavonols in our study. Because hesperetin and hesperidin were the only non flavonol aglycone and glycoside compound pair in our study, we were unable to establish any trend. However, the above result illustrates that the same change in metabolic composition could be interpreted to affect TAC very differently when evaluated using different TAC assays. It also indicates the need of comparative TAC studies using flavonoid-7-O-glucosides, although these compounds are difficult to obtain as test compounds.Flavonoids are generally present in plants as glycosides, and many as 3-wer TEAC  and DPPHwer TEAC ,50 than wer TEAC ,52. In o [p < 1% , confirmobserved ,54,55. Tobserved . In our glycones . This waycosides . The onlAnother example of various TAC assays evaluating the same change in polyphenol composition differently is the ratio of quercetin- and kaempferol-glycosides. The amounts of these compounds are strongly influenced by growth temperature and light conditions, although to different extents , and ulti.e. the presence of either hydrogen or O-methyl at this ring position) had a negative effect on FRAP or DPPH, while having no significant effect on TEAC or FCR. This is a new finding, and the fact that FRAP or DPPH assays give equally low  TAC for mono-hydroxy-benzoic acids and coumaric acids . Phenolic components were determined using HPLC-MS, and we found that leaves in year 2012 contained lower amounts of the same phenolic compounds than in the year 2013  were prepared according to the ratios of main phenolic components identified in leaf extracts as shown in O-glucuronide (Que-3-glu) or quercetin-3-O-glucoside (Que-3-glc) to contain an amount characteristic to 2013-mix. One aim of this experiment was to mimic an increase in one of the components only and see whether and to what extent antioxidant capacities were additive, i.e., whether such addition to 2012-mix was sufficient to increase antioxidant capacities to that of 2013-mix. We found that answers to these questions depended on the choice of assay. As shown in TAC parameters of these two mixtures were significantly different  and dimethyl sulfoxide. Synthetic leaf mimics were mixed from pure test compounds following molar ratios of major compounds identified in grapevine leaves by HPLC-MS .Pure test compounds for TAC measurements were purchased from Extrasynthase  and stored at 4 °C with the exception of apigenin, kaempferol-3-Vitis vinifera L. cv. Pinot noir were collected from the vineyard of the Institute of Viticulture and Oenology (University of Pécs), from Southern sides of East-West oriented rows. In both years, leaves were harvested at the onset of berry ripening (veraison), five leaves were pooled from each of three different plants and samples were lyophilized. Leaves were collected in two consecutive years, 2012 and 2013 of which the former was drier. For total FCR and TAC measurements, 150 mg of lyophilized leaf samples were extracted into 3 mL hydro-alcoholic solution , centrifuged at 13,000 rpm for 10 min in an Eppendorf minispin centrifuge  at room temperature. Supernatants were stored at −20 °C and used for TAC analyses. Leaves from 2CO3 (6% w/v) in microplate wells. After incubation at room temperature for 90 min, then absorbances were recorded at 765 nm using a Multiskan FC  plate reader. A calibration curve was made using Myricetin-3-O-glucoside and FCR values of test compounds were expressed relative to this flavonol. FCR of grapevine leaf samples was given as µM Myr-3-glc equivalent per mg leaf dry weight.Total phenol determination with Folin-Ciocalteu reagent  was appl•+) reduction was measured based on the method of Re et al. [•+ was prepared by mixing 0.1 mM ABTS, 0.0125 mM horse radish peroxidase and 1 mM H2O2 in a 50 mM phosphate buffer (pH 6.0). After 15 min, 10 µL diluted leaf extract or test compound was added to 190 µL ABTS•+ solution and conversion of the cation radical into colorless ABTS was followed as decrease in absorption at 651 nm. Instead of Trolox, Myr-3-glc was used to prepare a calibration curve and TEAC of test compounds were given in reference to that of Myr-3-glc. TEAC values of grapevine leaf extracts were expressed as µmol Myricetin-3-O-glucoside equivalents per mg leaf dry weight.2,2′-Azino-bis. For each sample, 10 µL diluted leaf extract or test compound was added to 190 µL freshly mixed FRAP reagent. Samples were incubated in microplate wells at room temperature for 30 min before measuring OD at 620 nm. FRAP values of grapevine leaf extracts were expressed as µmol Myricetin-3-O-glucoside equivalents per mg leaf dry weight, FRAP of test compounds were given in reference to that of Myricetin-3-O-glucoside.FRAP is based on detecting the capacity of samples to reduce ferric ions, which is measured as an absorbance change of ferrous TPTZ complex. The assay was carried out according to a modification  of the ov/v. For each sample, 20 µL diluted leaf extract or test compound was mixed with 180 µL of the above solution in a microplate well. After 30 min incubation in dark for 37 °C, the absorption change was measured at 517 nm and DPPH reducing capacities were given in reference to that of Myr-3-glc.The assay is based on the loss of violet color of DPPH  solution when reduced by an antioxidant . For the® Costar® Spin-X® plastic centrifuge tube filters , subsequently evaporated to dryness and resuspended in 200 µL of distilled water. Each extraction was carried out in duplicate.Lyophilized leaf samples were ground to fine powder and 0.02 g plant material was extracted three times in a final volume of 1.2 mL of 60% aqueous methanol for 90 min in total. The extract was filtered through Corning® Express F5 column  at a temperature of 30 °C using a water/acetonitrile gradient. Solvent A consisted of 99.5% water and 0.5% acetic acid; solvent B was 100% acetonitrile. The following gradient was used for Eluent B 5%–12% (0–3 min), 12%–25% (3–46 min), 25%–90% (46–49.5 min), 90% isocratic (49.5–52 min), 90%–5% (52–52.7 min) and 5% isocratic (52.7–59 min). The flow rate was 0.85 mL·min−1, and the detector wavelengths were set at 320, 330, and 370 nm. The hydroxycinnamic acid derivatives and flavonol glycosides were identified as deprotonated molecular ions and characteristic mass fragment ions by HPLC-DAD-ESI-MSn using an Agilent series 1100 ion trap mass spectrometer in negative ionization mode. Nitrogen was used as the dry gas  in addition to nebulizer gas (40 psi) with a capillary voltage of −3500 V. Helium was used as the collision gas in the ion trap. The mass optimization for the ion optics of the mass spectrometer was performed for quercetin m/z 301. The MSn experiments were performed in auto up to MS3 in a scan from m/z 200–2000. The standards, chlorogenic acid, quercertin-3-O-glucoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside and cyanidin-3-O-glucoside  were used for external calibration curves (0.01–10 mg per 100 mL).A HPLC series 1100 from Agilent  consisting of a degasser, binary pump, autosampler, column oven and photodiode array detector was used to determine the hydroxycinnamic acid derivatives and flavonol glycosides. The extracts were separated on a Supelco AscentisStatistical analyses of TAC data were performed using Excel Analysis ToolPack , and XLStat2006 . Graphs were prepared using SigmaPlot .R2 and p-values of t-tests. The null hypothesis of these tests was that the slope of the fitted regression line was zero. This hypothesis was rejected for data sets with p < 0.05 and these were concluded to be linearly correlated. Actual slope and intercept parameters are not given as these depend on the choice of the assignments of dependent/independent variables (unlike R2 and p) and the purpose of the calculation was to test connectivity between TAC rather than to give mathematical formulae to calculate one TAC from another.Pair wise relationships of data sets from different TAC methods were tested using simple linear regression. Linear fits were characterized by the regression coefficient p < 0.05 and, in this case, the factor was concluded to influence TAC.Effects of various structural aspects of the studied compounds on TAC values were evaluated using one-way ANOVA. In this analysis, a chosen structural feature was used as an independent nominal variable  and quantitative data from the chosen TAC measurement were used as a dependent variable. The null hypothesis was that the qualitative variable had no effect on the dependent quantitative variable. This was rejected for data sets with t-tests and TAC values of two data sets with p>0.05 were regarded as identical.TAC values of grapevine leaves were measured using three leaf extracts made from three sets of leaf samples (five leaves each) collected at the same time from different plants. All three leaf extracts were measured five times. In this way, standard deviations from mean values of the two leaf samples (year-2012 and year-2013) represent both biological variability and technical errors of TAC measurements. Synthetic mixtures of compounds were measured in triplicates and in these data sets standard deviations represent technical errors only. Differences between the two leaf samples and differences among synthetic mixtures were tested using paired


For successful autologous stem cell transplantation, the collection of a sufficient number of hematopoietic stem cells after induction therapy is essential for transplant candidates with multiple myeloma (MM).2 on day 1) or etoposide  in patients with MM. Granulocyte-colony stimulating factor  was administered from the onset of neutropenia to the final day of collection.In this study, we compared the efficacy and safety of stem cell mobilization using cyclophosphamide . The rate of mobilization failure, defined as < 2.0 × 106 CD34+/kg collected in three apheresis procedures, was lower in the VP-16 group than in the CY group . Severe infections during the mobilization period were more frequent in the CY group than in the VP-16 group .Sixty-five patients were mobilized with a combination of CY and G-CSF, and 63 were mobilized with a combination of VP-16 and G-CSF. All patients were mobilized within 7 months of beginning frontline treatment. The median number of CD34In conclusion, an intermediate dose of VP-16 with G-CSF appears to be an effective and tolerable chemo-mobilization method compared to CY and G-CSF, particularly in cases where use plerixafor in MM is difficult. P = 0.002). Furthermore, a recently published meta-analysis of three large phase 3 randomized controlled trials showed the superiority of upfront ASCT over novel agents [With the emergence of new and more effective therapeutic agents, the role of autologous stem cell transplantation (ASCT) in multiple myeloma (MM) has become a topic of considerable interest. Although there have been only a few studies evaluating the efficacy of ASCT with novel agent-based therapies, upfront ASCT did show a better response and a longer progression free survival (PFS) in a randomized phase 3 EMN02/HO95 trial  are the most common cell source for ASCT . As obta2 of VP-16 and G-CSF was highly effective and safe in MM patients. In their study, all patients achieved successful mobilization with a median number of CD34+ cells of 12 × 106/kg. Moreover, there were no reported collection failures, and no treatment-related mortalities. However, no studies have compared CY and VP-16 chemo-mobilization in MM patients. Therefore, we evaluated the efficacy and safety of stem cell mobilization using intermediate doses of VP-16 and high doses of CY in patients with MM.VP-16 is effective in mobilizing hematopoietic stem cells in patients with malignant lymphoma , 12. WooWe retrospectively analyzed the medical records of 140 patients diagnosed with MM who underwent stem cell mobilization between February 2008 and February 2018 at Chonnam National University Hwasun Hospital. We excluded patients who mobilized with G-CSF alone. Patients who were mobilized more than 12 months after initial treatment for delayed ASCT were also excluded. Following these exclusions, a total of 128 patients were included in this study. This study was approved by the Institutional Review Board of Chonnam National University Hwasun Hospital in accordance with the Declaration of Helsinki.2 on day 1) or VP-16 (375 mg/m2 on days 1 and 2) administration. G-CSF  was administered from the onset of neutropenia (absolute neutrophil count < 1 × 109/μL) up to the final collection day. For all patients, antimicrobial and antifungal prophylaxis was given using oral levofloxacin 500 mg/day and fluconazole 200 mg/day during the neutropenic period. Daily monitoring of circulating hematopoietic progenitor cells (HPCs), enumerated by the Sysmex XE2100 , was initiated at the start of leukocyte recovery, approximately 8 days after chemotherapy. Apheresis was initiated when HPC levels reached ≥5/μL, as described previously [Stem cells were mobilized with G-CSF following intravenous CY  was used to assess the clinical disease stage at diagnosis . The IntU-test, respectively. The PFS was calculated from the day of transplantation to disease progression or death from any cause. Overall survival (OS) was calculated from the day of transplantation to the day of death from any cause. PFS and OS were evaluated using the Kaplan-Meier method, and compared using the log-rank test. Estimates of the relative risk for an event and its 95% CI were obtained using the Cox proportional hazard model. All statistical analyses were performed using SPSS software . A P value < 0.05 was considered statistically significant.Discrete and continuous variables were evaluated using the chi-square test and a Mann-Whitney P <  0.001). Despite differences in frontline treatment, disease responses to the initial regimen before stem cell mobilization were similar in the two groups (P = 0.359). The median time from the first treatment to mobilization was shorter in the VP-16 group than the CY group  however, there were no differences in the median number of treatment cycles prior to mobilization . The number of patients who received spinal radiotherapy was similar in both groups .Between 2008 and 2018, a total of 128 patients with MM underwent stem cell mobilization and collection using CY or VP-16 within 12 months of their initial diagnosis. Sixty-five patients (50.8%) were mobilized with CY + G-CSF between February 2008 and December 2013, and 63 (49.2%) were mobilized with VP-16 + G-CSF between April 2014 and February 2018. The baseline clinical characteristics of both groups are summarized in Table P = 0.062). In the VP-16 group, only one patient showed mobilization failure and also failed to provide a sufficient amount of stem cells in a subsequent mobilization attempt using plerixafor. Among the seven patients who had mobilization failure with CY, three decided not to pursue ASCT, one received a bone marrow harvest, one underwent a repeated mobilization procedure with CY, and two received a second mobilization procedure with G-CSF only. The median number of days for leukapheresis was significantly shorter in the VP-16 group than in the CY group . The median number of CD34+ cells collected on day 1 was significantly higher in the VP-16 group than in the CY group . As a result, patients were transplanted with a larger number of hematopoietic stem cells in the VP-16 group than in the CY group .The mobilization data are summarized in Table P = 0.055). The median number of patients requiring platelet transfusion support during the transplantation period was fewer in the VP-16 group . There were no significant differences between the two groups with respect to the median time to neutrophil engraftment. The duration of post-PBSC infusion hospitalization was shorter in the VP-16 group.Fifty-seven patients (87.7%) in the CY group and 56 (88.9%) in the VP-16 group underwent upfront ASCT. The outcomes following ASCT are summarized in Table P = 0.806, 99.1 months vs. not reached, P = 0.113, Fig. After a median follow-up time of 84.0 months in the CY group and 12.6 months in the VP-16 group, PFS and OS were not significantly different between the groups . In addition, 47 (72.3%) patients in the CY group and 24 (38.1%) in the VP-16 group required platelet transfusions (P < 0.001). Non-hematological toxicities occurred in 35.4% of the CY group and 22.2% of the VP-16 group. Following mobilization chemotherapy, the most common non-hematological toxicity was infection. The overall incidence of infection after mobilization was higher in the CY group . Severe infection (≥ grade 3) also occurred more often in the CY group; however, this difference was not statistically significant . All patients who developed severe infections were successfully treated with broad spectrum antibiotics, which resulted in no patient deaths in either group. Other non-hematological toxicities included gastrointestinal and hepatotoxicity. One patient in the CY group, and one in the VP-16 group, showed a grade 2 aspartate transaminase/alanine transaminase elevation, which recovered following supportive care. One patient showed a mild hypersensitive reaction, including pruritus and facial swelling, after infusion of CY, which was resolved by chlorpheniramine injection. Two patients complained of grade 1 or 2 nausea and vomiting, and were also relieved of these symptoms after supportive care. There was no treatment-related mortality during the mobilization period in either patient group.More patients in the CY group required supportive transfusion during the mobilization period, which is defined as the time between the first day of mobilization chemotherapy infusion and the last day of collection. In all, 42 (64.6%) patients in the CY group and 25 (39.7%) in the VP-16 group needed transfusion with red blood cells . We surmise that these differences are due to disparities in the induction regimen schedule between the two groups. The majority of induction regimens at our institution follow a 28-day schedule; however, some regimens have a 21-day schedule. A greater number of patients in the VP-16 mobilization group were treated using the 21-day schedule regimen, although the number of induction cycles prior to mobilization were not different between the two groups. As a result, the median time to mobilization was slightly longer in the CY group than in the VP-16 group. A previous study on VP-16 mobilization in MM demonstrated that VP-16 successfully mobilized an adequate amount of stem cells (≥ 5.0 × 106 CD34+/kg) in patients who were previously given radiation therapy [+ cells collected was significantly different between the two methods. Although the number of patients evaluated was too small to warrant definitive conclusions, we speculate that the use of VP-16 is able to overcome the adverse effects of previous radiotherapy.In patients mobilized with VP-16, many more stem cells could be collected safely over fewer days of apheresis compared to CY mobilization. The number of patients for whom an adequate amount of stem cells could not be collected was also lower in the VP-16 group. The time from the first treatment to mobilization was also longer in the CY mobilization group than the VP-16 mobilization group (4.67 months vs. 3.88 months,  therapy . An evalIn this study, the incidence of infection was higher in the CY group than in the VP-16 group. Because of its highly immunosuppressive effect, CY has been used for the treatment of autoimmune diseases and acute or chronic graft-versus-host disease after allogenic hematopoietic stem cell transplantation. A significant decrease in the percentage of both central and effector memory T cells has also been reported in a murine model treated with CY . In a reThis study also included a patient in whom a sufficient number of stem cells failed to be collected after VP-16 mobilization. The subsequent use of plerixafor for immediate salvage was also unsuccessful. Plerixafor-containing regimens have around a 20% failure rate when implemented after failed mobilization, most likely as a result of a low or defective stem cell reserve , 22. FacBecause this study was retrospective in nature, the conditioning regimens in both groups differed significantly, and these differences might have influenced the transplantation results, such as time to neutrophil or platelet engraftment, and survival outcomes.+ cells collected from patients who were mobilized with an intermediate dose of VP-16 and G-CSF was significantly higher compared to those patients who were mobilized with CY and G-CSF. The mobilization failure rate was also lower in the VP-16 mobilization group than in the CY mobilization group. Although there were no statistically significant differences, severe infections during the mobilization period developed more frequently in the CY mobilization group. Therefore, an intermediate dose of VP-16 and G-CSF might be a more suitable chemo-mobilization regimen than a regimen of CY and G-CSF, particularly in situations where plerixafor cannot be implemented.In conclusion, the median number of CD34


The Patient Health Questionnaire-9 (PHQ-9) is a brief tool to assess the presence and severity of depressive symptoms. This study aimed to validate and calibrate the PHQ-9 to determine appropriate cut-off points for different degrees of severity of depression in Argentina.We conducted a cross-sectional study on an intentional sample of adult ambulatory care patients with different degrees of severity of depression. All patients who completed the PHQ-9 were further interviewed by a trained clinician with the Mini International Neuropsychiatric Interview (MINI) and the Beck Depression Inventory-II (BDI-II). Reliability and validity tests, including receiver operating curve analysis, were performed.p < 0.01)]. For the diagnosis of Major Depressive Episode (MDE) according to the MINI, a PHQ-9 ≥ 8 was the optimal cut-off point found . The local version of PHQ-9 showed good ability to discriminate among depression severity categories according to the BDI-II scale. The best cut off points were 6–8 for mild cases, 9–14 for moderate and 15 or more for severe depressive symptoms respectively.One hundred sixty-nine patients were recruited with a mean age of 47.4 years (SD = 14.8), of whom 102 were females (60.4%). The local PHQ-9 had high internal consistency  and satisfactory convergent validity with the BDI-II scale [Pearson’s correlation = 0.88 (The Argentine version of the PHQ-9 questionnaire has shown acceptable validity and reliability for both screening and severity assessment of depressive symptoms. Major Depressive Episodes (MDE) are one of the leading causes of the global disease burden . In seveIt has been estimated that about 20% of adults in low and middle-income countries (LMIC) suffer from mental health or substance use disorder each year . In LatiThere is a variety of available instruments to assess depressive symptoms, but most of them have been developed in high-income countries and have not been cross-culturally adapted or validated for their use in LMIC . The ninThe PHQ-9 is a short, self-administered questionnaire, widely used for screening of depression in primary care settings , and detAlthough there is a cross-culturally adapted version of the PHQ-9 in Spanish for Argentina , this veA cross-sectional analytical study was conducted on adults with different degrees of severity of depression as well as individuals with no depressive symptoms. The study sample was obtained between December 2013 and March 2014. Patients were recruited from two primary care clinics and two specialty mental health outpatient facilities, both from the City of Buenos Aires, Argentina. The out-patient facilities were: 1) the “Dr. Braulio A. Moyano” Hospital, which is a public neuropsychiatric hospital serving a large urban catchment area predominantly of low-income, uninsured patients; and 2) the “Foro Foundation”, a private outpatient facility treating high-income patients. The primary care clinics were: 1) The “Cooperativa de Grupo de Práctica de Medicina Familiar”, a private primary care center that treats middle-income insured patients from anywhere in Buenos Aires; and 2) the “Centro HORUS”, a private institution specialized in mental health with a multidisciplinary approach serving middle-income patients.A purposeful quota sampling approach of persons attending these facilities was used in the study. Participants were recruited from two sources: 1) Patients referred by physicians because of the previous diagnosis of depression, and 2) patients who asked for an appointment for other health problems were approached and invited to participate. In both cases, all patients were invited to participate and asked for their signed informed consent.Beck Depression Inventory described below. A minimum of thirty patients per category was set for quota sampling.Patients were recruited until the fulfillment of four quotas defined as follows: , according to the Patients were included if they were able and willing to consent, aged 21 years or older, and were native speakers of Spanish. Exclusion criteria only applied to patients who were illiterate.We used the existing Argentinian version of the PHQ-9 instrument, which went through a full cross-cultural adaptation process .This is a nine-item self-reported scale, developed to diagnose the presence and severity of depressive symptoms in primary care and the community. It is based on the DSM-IV diagnostic criteria for Major Depression Episode and it has the potential to be a dual-purpose instrument that can establish a tentative diagnosis of a depressive episode as well as depressive symptoms severity . Each quIn the present study, we will validate and calibrate the PHQ-9 as a continuous measure.The continuous measure is a summary score ranging from 0 to 27 and is calculated by adding up the responses to the nine questions, which allows assessing the presence and severity of a depressive episode . The iniThe Spanish version of the Mini International Neuropsychiatric Interview (MINI)  was usedThe locally validated version of the Beck Depression Inventory Second edition (BDI-II) was used as an instrument to ascertain symptom severity . The BDIWe decided to address the inherent difficulty given by the fact that PHQ-9 defines five categories of depression while the BDI-II defines only four, because to our knowledge BDI-II was the unique instrument for depression screening validated in Argentina at the beginning of the study. So, in our study, the moderately severe and severe categories of the original PHQ-9 were expected to correspond to the severe category of BDI-II.The PHQ-9 was self-administered, while a trained clinician conducted a structured interview  and applied the BDI-II questionnaire. Only those individuals who did not meet criteria for MDE received the Dysthymia module, as we wanted to ascertain how many of those classified as ‘no depressed’ could present low levels of depressive symptomatology. To minimize a possible response bias induced by the sequence of administration of the instruments, two random sequences were used as follows: a) MINI, BDI-II and PHQ-9, and b) PHQ-9, MINI, and BDI-II. All the clinicians who conducted the interviews were blinded to the results of the PHQ-9.Additionally, we collected information on age, gender, level of education, marital status, employment, and health coverage.Considering expected values of sensitivity between 85 and 88%, and specificity between 92 and 95%, we calculated a minimum sample size required of 40 participants for each level of severity of depression and 30 healthy subjects with no depressive symptoms. For the sample size calculation we used the “Epidat 4.1”, free statistical software developed by Dirección Xeral de Innovación e Xestión da Saúde Pública de la Consellería de Sanidade  and funded by PAHO and WHO.Criterion validity was evaluated through the comparison of the scores obtained with the PHQ-9 with the MINI interview for diagnosis, and BDI-II for the severity of depression. We calculated sensitivity, specificity and positive predictive value (PPV) and negative predictive value (NPV).To determine the most appropriate cut-off points for PHQ-9 receiver operating characteristics (ROC) curves were generated and Youden index was calculated using the PHQ-9 summary score, where the results for depression diagnosis and severity were obtained from MINI and BDI-II respectively. All estimates were given with 95% confidence intervals.To determine the optimal cut-off points, the area under the curve (AUC) and the PPV and NPP were evaluated and compared to the original cut-off points suggested by the authors of the original scale . The AUCThe data were analyzed with dysthymia cases included as “not depressed” and also excluding them to evaluate eventual changes in the results.SD 14.8 years). Thirty-eight percent of them were secondary school graduates, and 14.8% were unemployed. Thirty percent of participants were married or had a partner, and 77% had social or private health insurance (Table SD = 13.4) with a median score of 20 points (IQR = 19).A total sample of 169 subjects was recruited, 102 women (60.4%) and 67 men (39.6%). The mean age was 47.4 (%) met the diagnosis of DSM-IV MDE. The mean PHQ-9 score for these patients was 14.76 (SD = 5.65), whereas the mean score for patients without diagnosis of MDE was 4.16 (SD = 4.01).We examined the performance of PHQ-9 against the diagnosis of MDE by MINI as the gold standard. According to MINI, 102 patients (60.36% of subjects were correctly classified. An area under the curve (AUC) of 0.87  also suggests good accuracy.  of dysthymia.We analyze the data with dysthymia cases included as “not depressed” first and excluding them from the analysis subsequently but the results were unaltered either way, most likely because there were only few cases (p < 0.01) indicating a positive and strong correlation between both instruments. , 0.88 (95% CI 0.83 to 0.93) and 0.86 (95% CI 0.80 to 0.92) respectively  and specificity (90.4%) and yielded a Youden index of J = 0.82 that represented 91.12% of subjects correctly classified. When comparing AUC for a cut-off point of 6 and for a cut- off point of 5  the difference was not statistically significant (CI overlapped). A cut-off point of 5 showed an AUC 0.85 (95% CI 0.79–0.91) whereas a cut-off point of 6 showed an AUC of 0.91 (95% CI 0.86–0.95).Regarding moderate symptoms of depression, at the cut-off point of 9, the sensitivity was high (95.3%) but the specificity was lower but still adequate (81.0%) and the Youden index was J = 0.76. This classification yielded 88.17% of subjects correctly classified. When comparing AUC for a cut-off point of 9 and for a cut- off point of 10  the difference was not statistically significant (CI overlapped). A cut-off point of 9 showed an AUC of 0.88 (95% CI 0.83–0.93) whereas a cut-off point of 10 showed an AUC of 0.87 (95% CI 0.82–0.92).CI 0.80–0.92) and cut-off point of 20 showed AUC 0.67 (95% CI 0.60–0.74). Optimal cut-off points for the Argentinian version of PHQ-9 are shown in Table Finally, the best cut-off point to measure severe depressive symptoms was 15 or higher, with a sensitivity of 82.4% and a specificity of 89.0%. Using that cut-off point, we obtained a Youden index of J = 0.71 and the PHQ-9 questionnaire correctly classified 86.98% of the subjects. In this case, as a comparison of both ROC curves for the cut-off point of 15 and the cut- off point of 20  a significant difference was obtained. Cut-off point of 15 showed AUC 0.86  determined the standard diagnostic practice. While the use of Dysthymia module of the MINI (time frame of two years) just helped us to capture the patients with lower levels of depressive symptomatology but who do not reach MDE criteria. As we explained before, only those individuals who did not meet criteria for MDE received the Dysthymia module. We found that including or excluding these patients did not alter the results at all, something that was expected as there were few cases of dysthymia.Of note, the PHQ-9 score was highly correlated with the BDI-II score. This correlation was even higher than that reported by Kneipp et al. (Pearson Correlation Coefficient = 0.80) when comparing the same instruments in low-income female populations . Our resRegarding the comparison of categories of severity and despite the inherent difficulty given by the fact that PHQ-9 defines five categories of depression while the BDI-II defines only four, the optimal cut-off points for the Argentine version of PHQ-9 generated the same four categories, as found in other studies (see Table This validation study of the PHQ-9 for the Argentinean population has several strengths. First, it was rigorously designed to have an adequate representation of all the stages of severity of depression, including patients with non-depressive symptoms, which is key to ensure not only its validity for diagnosis of depression but also its calibration for different categories of severity; Secondly, we chose a criterion tool that is shorter than other diagnostic tools available in Argentina for identifying depressive cases , somethiOur study presents some limitations. First, since this study has focused mainly on the city dwellers of Buenos Aires and its surroundings, its extrapolation to rural settings should be taken with caution. Yet, its usability seems to be enhanced by the fact that 90% of the Argentine population lives in urban areas. Second, the sample composition is heterogeneous with patients coming from primary and secondary care settings as well as private and state sectors. Nonetheless, this might allow us to extrapolate findings to other clinical populations. Third, we administered the instruments in a different order to avoid eventually bias, but we have not done additional analyses to ascertain if this had an impact on results. Fourth, since the PHQ-9 defines five categories of depression while the BDI-II defines only four, the optimal cut-off points for the Argentine version of PHQ-9 generated the same four categories. However, it may not have a relevant impact on screening or intervention purposes because it doesn’t condition the therapeutic approach.The Argentine version of the PHQ-9 questionnaire has shown acceptable validity and reliability for both screenings of Major Depressive Episodes and severity assessment of depressive symptoms. A definite diagnosis would ideally be attained; however, with a complementary psychiatric interview; a tool that is not always available in primary care settings. Therefore, this validated and calibrated tool could improve and facilitate the detection, classification and monitoring of depressive disorders in Argentina, particularly in the primary care setting, where depression still goes unnoticed and therefore undertreated.


The traditional Chinese formula Da-Cheng-Qi-decoction (DCQD) has been used to treat acute pancreatitis for decades. DCQD could ameliorate the disease severity and the complications of organ injuries, including those of the liver and lungs. However, the pharmacological effects in the kidney, a target organ, are still unclear. This study aimed to investigate the herbal tissue pharmacology of DCQD for acute kidney injury (AKI) in rats with severe acute pancreatitis (SAP).Rats were randomly divided into the sham-operation group (SG), the model group (MG) and the low-, medium- and high-dose treatment groups . Sodium taurocholate (3.5%) was retrogradely perfused into the biliopancreatic duct to establish the model of SAP in rats. Different doses of DCQD were administered to the treatment groups 2 h after the induction of SAP. The major components of DCQD in kidney tissues were detected by HPLC–MS/MS. Inflammatory mediators in the kidney tissues, as well as serum creatinine (Scr), blood urea nitrogen (BUN) and pathologic scores, were also evaluated.p < 0.05). The high dose of DCQD showed a maximal effect in downregulating the pro-inflammatory mediators interleukin-6 (IL)-6 and tumour necrosis factor-α (TNF-α), upregulating anti-inflammatory mediators IL-4 and IL-10 in the kidney and alleviating the pathological damages. DCQD decreased the pancreas and kidney pathological scores of rats with SAP, especially in the HDG (p < 0.05). Compared with the MG, the level of Scr in the HDG was significantly decreased (p < 0.05).Ten components of DCQD were detected in the kidneys of the treatment groups, and their concentrations increased dose-dependently. Compared with the SG, the levels of inflammatory mediators, Scr, BUN and pathological scores in the MG were obviously increased  contains supplementary material, which is available to authorized users. Acute pancreatitis (AP) is commonly a self-limited inflammatory disease caused by abnormally activated pancreatic digestive enzymes . However2 (PLA2), hypoxemia, as well as a decrease in renal perfusion pressure due to abdominal compartment syndrome and an impairment of renal microcirculation for the release of pancreatic amylase, may also play important roles in the pathophysiology of SAP-induced AKI [The mechanism of SAP resulting in AKI is complex. Available studies revealed that SAP-induced AKI is mainly related to SIRS , which iuced AKI . The moruced AKI . Althouguced AKI , 14, theuced AKI  other thuced AKI . Howeveruced AKI . A multiuced AKI . Hence, Rheum palmatum L. (Dahuang), Magnolia henryi Dunn. (Houpu), Citrus aurantium L. (Zhishi) and Natrii Sulphas (Mangxiao), has been applied to treat AP effectively for decades in China [Da-Cheng-Qi decoction (DCQD), which is composed of in China . We hypoin China –22. HoweThe information regarding the experimental design, statistics, and resources used in this study are attached in the minimum standards of reporting checklist  were purchased from Chengdu Dashuo Bio-Technique Co. Ltd. . The experimental protocol was performed according to the Animal Ethics Committee Guidelines of our hospital . One week after acclimation, the animals were fasted with free access to water for 24 h prior to the experiment.Treaties on Exogenous Febrile Disease, the suggested dose of DCQD for a person weighting 60 kg is 57 g, composed of 12 g of Dahuang, 24 g of Houpu, 12 g of Zhishi and 9 g of Mangxiao. As we have mentioned [The spray-dried drug powders were obtained from Chengdu Green Herbal Pharmaceutical Co. Ltd. . The processing procedure of the crude formula components has been previously described . Accordientioned , we chosThe rats were randomly divided into five groups and marked as the low-dose treatment group , the medium-dose treatment group , the high-dose treatment group , the sham-operation group (SG) and the model group (MG). The SAP model was induced via biliopancreatic duct injection of 3.5% sodium taurocholate  with a micro-infusion pump at a rate of 0.2 mL/min (1 mL/kg body weight) . The ratTwenty-four hours after administration, all the rats were sacrificed, and arterial blood as well as kidney pancreas tissues were collected for measurement. Pancreas and kidney tissues were cut into slices and were fixed with 10% neutral formalin, embedded in paraffin, cut into sheets (at thickness of 5–7 μm) and then stained with haematoxylin and eosin. Pathological scores were blindly evaluated by two independent pathologists with a previously established scoring system , 22, 23.Additional pancreas and kidney tissues were stored at − 80 °C. High-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was used to measure the major components of DCQD  in the kidney tissue homogenates (10%) . As we dThe levels of IL-6, TNF-a, IL-10 and IL-4 in kidney tissue homogenate were measured by using the Milliplex MAP Rat Cytokine/Chemokine magnetic bead immunoassay kit . The blood samples were centrifuged at 3000 rmp for 5 min and the serum was distracted for Scr and BUN. The concentrations of Scr and BUN were detected by an Automatic Biochemical Analyser .The ten major components of DCQD from the previous study , 24 were2. Concentrations of QC and unknown samples were measured by interpolation from the calibration curves [Data collection, peak integration, and calibration were all calculated with Analyst 1.4.2 software. Calibration curves were plotted according to the peak ratio of analytes to internal standards (ibuprofen), and the linear regression between tissue concentrations and peak area ratios were determined by 1/χn curves .p < 0.05.The statistical analysis was performed with PEMS3.1 for Windows . All the data were expressed as the mean ± standard deviation (mean ± SD). One-way repeated-measures ANOVA, followed by multiple pair-wise comparisons using the Student–Neuman–Keuls procedure, was applied to the analysis of multiple groups. The data was considered significantly different when The ten major components of DCQD were all detected in the kidney tissues. The concentrations of emodin, rhein, aloe-emodin, chrysophanol, rheochrysidin and magnolol increased with the DCQD dose and showed significant differences when compared to the other treatment groups. The concentrations of hesperidin, naringin, naringenin, and honokiol were not as closely related to the dose. Rhein and naringenin were relatively higher than the other compounds of DCQD in all the treatment groups Fig. .Fig. 1Kip < 0.05), but there was no change in IL-10. Compared to the MG, the pro-inflammatory mediators in all the treatment groups were downregulated (p < 0.05), while the anti-inflammatory mediators in all the treatment groups were upregulated obviously (p < 0.05). The lowest level of pro-inflammatory mediators and the highest level of anti-inflammatory mediators were both in the HDG  and anti-inflammatory mediators (IL-4) in the MG were significantly increased compared to those in the SG . The level of Scr in the HDG was significantly lower than that in the MG (p < 0.05), without a difference in BUN  decreased and the anti-inflammatory mediators (IL-4 and IL-10) increased along with the dose. Moreover, the pathological scores of the kidneys and pancreas showed the same tendency. The highest dose of DCQD had the maximal effects in downregulating the pro-inflammatory mediators, upregulating anti-inflammatory mediators in the kidney and ameliorating pathological damage. The data demonstrated the regulatory effects of DCQD in the inflammatory response to ameliorate AKI with SAP and to eventually attenuate the severity of SAP. Our previous studies revealed a similar effect of DCQD on damage to the lungs, pancreas, intestine and liver [In our study, we detected the levels of TNF-cated AP . After gnd liver –22. Zhaond liver  reportednd liver  found thnd liver . Rhein ind liver  and to pnd liver . The narnd liver  and to rnd liver . Li et and liver . Future In 1998, Zhao et al.  came to In conclusion, most of the major components of DCQD were absorbed into the kidney of rats with SAP and their concentrations increased dose-dependently. Above all, DCQD ameliorated AKI in rats with SAP by regulating the inflammatory response, and it might be closely related to the intake dose.Additional file 1. The minimum standards of reporting checklist.


In clinical conditions, orthodontic brackets are exposed to periodic stresses mainly induced by mastication and intraoral forces. The objective of the present study was to evaluate the effects of cyclic loading to simulate masticatory forces on shear bond strength (SBS) of metal brackets bonded to teeth using self-etch and total-etch bonding systems.Eighty-four caries- and crack-free bovine mandibular incisors were selected and randomly assigned to two groups based on the type of bonding system. After bonding, all samples were thermocycled (500 cycles) followed by cyclic loading of the half of the specimens in each group by applying 40 N load with 2 Hz frequency for 10,000 cycles. The SBS was measured using a universal testing machine. The adhesive remnant index (ARI) score was calculated subsequently. Data were analyzed using Kolmogorov-Smirnov test, two-way ANOVA and Mann-Whitney test.The SBS was 10.09±3.78 MPa and 14.44±6.06 MPa for self-etch and total-etch bonding systems in cyclic loading group, respectively. The SBS was 9.43±5.3 MPa and 11.31±5.42 MPa in self-etch and total-etch groups without cyclic loading, respectively. Cyclic loading did not cause any significant difference in SBS (P>0.05). The ARI scores of the groups were significantly different (P<0.05).The present results demonstrated that low masticatory forces at 10,000 cycles did not have a significant impact on bracket-adhesive SBS; however, they significantly changed the ARI score. Even though the total-etch bonding system yielded higher SBS than the self-etch system, the performance of both was clinically acceptable. Optimal bracket-adhesive-enamel bond strength is essential for reliable transfer of forces from arch-wire to teeth. A number of factors, other than the bonding agents and brackets, can influence the bond strength of brackets to teeth. Among these factors, intraoral forces are of great significance . MasticaIn the present study, 84 extracted bovine mandibular incisors were selected and disinfected by immersion in 0.5% chloramine T solution at 4°C for one week after removing the soft tissue remnants and debridement. The teeth were caries-free and had no apparent defects or enamel damage. Although bovine and human teeth are different, studies have shown that there is no significant difference in adhesive bond strength to enamel between the two ,13. The 2 were bonded to bovine incisors with Transbond XT light-cure composite  following the application of bonding agent. Before curing, excess composite resin was removed with an explorer and then the resin was cured for 20 seconds, 10 seconds mesially and 10 seconds distally, using a light-emitting diode light-curing unit . All samples were prepared and bonded by one operator, and 300 g force was applied during bracket bonding using a force gauge . After bonding, all samples were stored in distilled water at 37°C for 24 hours. Subsequently, the samples were thermocycled for 500 cycles between 5°C and 55°C with a dwell time of 60 seconds. The transfer time between baths was 8 seconds.Stainless-steel upper right central incisor brackets  with a base surface area of 12.68 mm2).After thermocycling, all samples were mounted in auto-polymerizing acrylic resin such that the bonding surface was perpendicular to the horizontal plane. For this purpose, after bonding of samples, a straight 25x21 stainless steel wire was placed in the bracket slot to ensure perpendicular position of bonded surface relative to the horizontal plane. Afterward, each group was divided into two subgroups; one subgroup was subjected to cyclic loading  ,14, 15 wFollowing debonding, the enamel surface of each tooth and the bracket base were inspected under a stereomicroscope  at ×10 magnification.The modified ARI scores  were calKolmogorov-Smirnov test confirmed normal distribution of data. Thus, the collected data were subjected to descriptive statistics. Two-way ANOVA was used to determine the effect of cyclic loading and adhesive system on SBS. The Mann-Whitney U test determined the differences in ARI scores among the groups. Significance for all statistical tests was determined at P<0.05, and SPSS version 15.0 was used to analyze the data.Two-way ANOVA indicated that the effect of cyclic loading on SBS was not significant (P=0.1). The interaction effect of cyclic loading and the adhesive system on SBS was not significant either (P=0.28), but the type of adhesive system had a significant effect on SBS (P=0.008). The SBS was significantly higher in TE-N compared to SE-N group . In samples subjected to cyclic loading, the mean SBS was lower in SE-C group (11.92±5.39 MPa); however, this difference was not significant (P=0.101). Comparing SBS before and after cyclic loading revealed no significant difference . The Mann-Whitney U test revealed a significant difference between the subgroups of each bonding agent in ARI score (P<0.0001); in SE-N group, more than 50% of samples had a score of 5, which indicates obvious fracture on the enamel surface while after cycling loading only 28.6% of samples had a score of 5.Opposite results were observed in the total-etch group; 52.4% of samples had a score of 1, which means most of the adhesive remained on the tooth surface (and less than 25% of adhesive remained on the bracket base); whereas, after cyclic loading, less than 5% of samples showed a score of 1. Comparing the ARI scores between TE-N and SE-N showed a significant difference between the two groups (P=0.002); however, after cyclic loading, the difference was not significant (P=0.44). The aim of the present study was to evaluate the effect of cyclic loading on SBS of metal orthodontic brackets bonded to enamel surfaces with two commonly used adhesive systems.In measuring the SBS of brackets to tooth structure, the blade technique has shown good reproducibility . MoreoveIn the present study, 40 N force was applied to bonded brackets since it is the lowest amount of force that is reportedly applied to orthodontic appliances during mastication . Also, sMost similar studies determined fatigue strength by applying 1000 cycles and compared it to static SBS ,3. The aAlthough certain aspects of physical and mechanical properties of bonding systems can be clarified by in vitro experiments, it is necessary to simulate clinical conditions in laboratory testing as much as possible. In the current study, 10,000 cycles were applied because this was the highest frequency of cyclic loading submitted during fatigue testing , 14. It The minimum acceptable bond strength of brackets is between 6–8 MPa . MoreoveThe mean SBS of the self-etch adhesive system was lower than that of the total-etch group; nonetheless, it was still within the clinically acceptable range. There is some controversy in comparison of total-etch and self-etch adhesives. Although a number of studies have shown a higher value of SBS in total-etch groups ,27, someIt has been shown that it is more desirable to have resin remnants on the enamel surface after bracket debonding to decrease the risk of enamel damage . In thisIn order to more realistically simulate the clinical conditions, further studies with higher load and higher frequency of cycles are required to provide more detailed information in this respect.The results of the present study demonstrated that low masticatory forces of 40 N at 10,000 cycles could not cause a significant difference in bracket-adhesive SBS; however, they could significantly change the ARI score. Although the mean SBS of total-etch system was higher than that of self-etch system, this difference was no longer significant after cyclic loading, and all values were within the clinically acceptable range. It seems that self-etch bonding systems are superior to total-etch systems for routine use in orthodontic clinics considering their optimal bond strength as well as other advantages of self-etch systems.